This invention relates to coupling agents useful in the production of biologically useful molecules labeled with metal ions. The ions may be either radiolabels or paramagnetic ions. Radiolabeled antibody fragments incorporating the coupling agents of the invention are useful in therapeutic and in vivo diagnostic applications. Paramagnetic labeled biologically useful molecules incorporating the coupling agents of the invention are useful in in vivo diagnostic applications.
Paramagnetic metals capable of influencing NMR relaxation properties for image enhancement have been known for some time, for example, Gd(III), Mn(II), Fe(III) and Cr(II).
The efficacy of paramagnetic metal ions in in vivo diagnostic applications depends on the ability to deliver the metal to the target site and have it influence the relaxation of the water in that region in an NMR imaging experiment.
The use of radionuclide metal ions in therapeutic and in vivo diagnostic applications has been practiced for some time. For example, gamma-emitting radionuclide metal ions, such as indium-111, gallium-67 and technetium-99m, have been used in diagnostic scintigraphy for tumor detection. Beta-emitting isotopes, such a rhenium-186, rhenium-188, rhenium-189, samarium-153, yttrium-90 and copper-67, can be used therapeutically in the treatment of tumors.
The efficacy of radionuclides in in vivo diagnostic and therapeutic applications depends on the ability to deliver the radionuclide to the site of the target cells. One method of delivering the radionuclide to the site of the target cells entails coupling the radionuclide metal ions to biologically useful molecules, such as antibodies, which selectively recognize and bind unique ligands associated with the target cells. For example, antigens which are known to be produced by or associated with malignant tumor cells can be bound by the antibody-conjugated radionuclide for the purpose of diagnostic imaging or for the purpose of irradiating the tumor to destroy it.
Goldenberg et al. (N. Engl. J. Med., 298: 1384-1388 [1978]) describe experiments in which antibodies to carcinoembryonic antigen (CEA), which is a known tumor-associated antigen (Gold et al., J. Exp. Med., 121: 439-462 [1965]), were labeled with iodine-131 and injected into patients with a history of cancer. After 48 hours, the patients were scanned with a gamma scintillation camera and tumors were localized by the gamma emission pattern. Similarly, United Kingdom Patent Application GB No. 2,109,407 describes the use of monoclonal antibodies to tumor-associated antigens, labeled with metallic radionuclides, for in vivo tumor detection and localization.
It has been suggested that radiolabeled antibody fragments, rather than radiolabeled whole antibodies, be used for in vivo diagnostic and therapeutic applications since the fragments may be better able to penetrate to the desired target site and the antibody fragments may minimize problems of immunogenicity and cross-reactivity associated with whole antibodies (see, e.g., U.S. Pat. No. 4,036,945; Lancet, Vol. II, No. 8087, 462 [1978]; Belitsky et al., J. Nucl. Med., 19: 429 [1978]). Antibody fragments can be produced in several ways. The antibody molecule can be enzymatically treated to remove carboxyl-terminal terminal portions of the heavy chains (the Fc fragment), leaving a bivalent F(ab').sub.2 fragment, i.e., two Fab' segments joined by one or more disulfide bonds which link the heavy chains. The F(ab').sub.2 fragment can then be selectively reduced at the disulfide bond(s) joining the two heavy chains, resulting in the production of two monovalent Fab' fragments each having a single antigen-binding site.
Antibody molecules contain a number of reactive side chains which can be employed as sites of attachment for binding a radionuclide metal ion to the antibody. For example, the radionuclide can be conjugated to the antibody through a linker molecule which is reactive with the carboxyl groups of aspartic acid or glutamic acid residues, the amino groups of lysine residues or the aromatic groups of tyrosine or histidine. Unfortunately, these residues are distributed randomly throughout the antibody molecule. Consequently, attachment of a radionuclide through these reactive groups can occur at any of a number of points along the antibody molecule. Attachment of the radionuclide-bearing moiety at or near the antigen binding site on the antibody molecule could inhibit the ability of the antibody to bind antigen, resulting in a loss or reduction of the effectiveness of the antibody-radionuclide conjugate as a diagnostic or therapeutic agent. There is needed a method of attaching a radionuclide to an antibody which does not have a significant adverse affect on the immunoreactivity of the antibody.